DESCRIPTION:The goal of this proposal is to define the genetic network that coordinates the initiation of DNA replication with cell cycle progression. Caulobacter crescentus is particularly well suited to the identification and analysis of the regulatory factors that mediate replication initiation and chromosome movement in a simple bacterial cell with a well-defined cell cycle. The unique characteristics of Caulobacter include ease of cell synchrony, one and only one replication of the single chromosome per cell cycle, a clear G1, S, and G2 period, and an asymmetric division that yield progeny with distinct morphological features and different cell fates. The initiation of DNA replication is under the negative control of the CtrA response regulator which binds to the replication origin. CtrA is temporally controlled by phosphorylation and proteolysis, and is asymmetrically restricted to the non-replicating progeny swarmer cell. In addition, expression of the genes encoding the replication machinery are induced at the time of replication initiation and coordinately regulated, and a new DNA methylating enzyme, CcrM, contributes to the control of replication initiation, providing yet other layers of regulation. The proposed experiments will seek to coordinate these layers of control with the factors that control cell cycle induction of the genes encoding the replication machinery, determine the role of DNA methylation sites in the replication origin, and define the regulatory mechanisms that confine the CcrM DNA methyltransferase to a specific time period late in the cell cycle. Components of the chromosome segregation apparatus will also be identified and how they are cued to the progression of chromosome replication will be determined.